This Phase I SBIR proposal targets an unmet critical need in Process Analytical Technology (PAT), the on-line quantitation of cell density during the development and manufacture of biopharmaceuticals. A non-destructive, optical method is proposed that operates continuously and in real-time during production of biologics in yeast and mammalian cell-based bioprocesses. This approach leverages an existing innovative Bioprocess Monitor that automatically measures the concentration of key chemicals during production in yeast (Pichia pastoris) and mammalian cell (CHO) expression platforms. Using near-infrared spectroscopic methods, this measurement system has demonstrated unprecedented robustness in bioprocess chemical quantitation, including industrial operations over a year without need for additional calibration. A research plan is proposed to establish the technical and commercial feasibility of quantifying cell density measurements (cells/mL) using this Bioprocess Monitor for Pichia pastoris and CHO cell platforms. Specific Aims for this proposal include 1. Evaluation of critical parameters affecting cell density quantitation and alternative measurement combinations 2. Assessment of calibration robustness during live protein expression bioprocesses. In addition to the high value of this measurement by itself, the combination of quantitative cell density with key nutrient and metabolite concentrations opens an impressive range of fundamental and engineering opportunities. For example, fundamental measurements of nutrient/metabolite utilization per cell can now be evaluated and optimized in real-time during the complex bioprocess operation. Significant advances in product yield as well as product consistency are anticipated as a result of this technology, decreasing the cost and time to market for these complex and valuable medicines.